石蜡包时组织的DNA提取及其在免疫球蛋白基因重排…

基因重排分析在淋巴瘤诊断中具有重要意义。文章应用改良ＤＮＡ提取方法。从３０例淋巴生性病变石蜡包埋组织获得的ＤＮＡ虽有不同程度的降解,但适于ＰＣＲ扩增Ｉｇ重链箕因重排分析;约１／３病例提出高分子量ＤＮＡ,可用于ＤＮＡ印迹杂交。因此,石蜡包埋组织同样可为某些疾患,如淋巴瘤凝难和罕见病例的回顾性分子病理学研究提供基因诊断的ＤＮＡ来源。     

N-甲基亚硝基脲诱导小鼠胸腺淋巴瘤TCR基因重排分析

目的探讨N-甲基亚硝基脲（MNU）诱导的小鼠胸腺淋巴瘤的单克隆起源。方法采用巢式PCR方法,对8例MNU诱导的胸腺淋巴瘤组织进行T细胞受体β链（TCRβ）和γ链（TCRγ）克隆性基因重排分析,并对TCRγ基因重排的PCR产物直接测序。结果 8例胸腺淋巴瘤检测TCRβ和TCRγ均呈克隆性基因重排。DNA序列测定证实TCRγ基因PCR扩增产物为基因重排产物。结论巢式PCR TCR基因重排检测及DNA序列分析证实,MNU诱导的小鼠胸腺淋巴瘤是来源于T细胞的肿瘤。     

从石蜡包埋组织中获取高质量基因组DNA的方法改进

从石蜡包埋组织中制备基因组DNA(gDNA)难度大、获取样本量很少.传统方法为二甲苯脱蜡法.为了简化操作,获得更高质量的gDNA,我们采用水浴法替代二甲苯进行脱蜡,亲和层析纯化石蜡包埋肝细胞癌(HCC)组织标本中基因组DNA,以纯化gDNA为模板PCR扩增看家基因.结果显示改良水浴法提取的gDNA质量、数量均显著高于传统二甲苯脱蜡法,PCR产物量也得到显著提高.与传统方法相比,改良水浴法简单、快捷,可以更有效的回收石蜡包埋组织中的gDNA.     

石蜡包埋组织中DNA的提取

从石蜡包埋组织中提取DNA,是进一步研究抑癌基因的基础,而石蜡包埋组织中提取到的DNA往往已被降解成小分子且量微,作者经多次摸索试验,改进了一些步骤,取得了较好的抽提效果。     

从石蜡包埋和甲醛浸泡组织中提取DNA进行PCR扩增的研究

如何从石蜡包埋组织中高质量的提取DNA从而进行分子生物学特别是基因工程方面的科研工作,目前在国内尚未见报道。我们采用二甲苯或水浴法对2块胃癌石蜡包埋组织进行脱蜡,并提取、纯化了DNA,进行PCR扩增,获得了成功。另外对用甲醛浸泡的组织其DNA的受损程度与上述进行了比较,现报告如下。     

Bst DNA聚合酶大片段在全基因组扩增中的应用

Bst DNA聚合酶大片段作为一种常用的DNA聚合酶,因其独特的特点:能引发链置换反应、高保真、耐高温等,而成为一种重要的DNA多重置换扩增酶。目的:为减少成本,设计一种高产,方便且扩增活性高的Bst DNA聚合酶大片段表达体系;探究该酶应用于胃癌石蜡包埋组织基因组DNA的扩增条件。方法:采用p TWIN1质粒作为载体克隆表达Bst DNA聚合酶大片段,应用几丁质亲和层析柱纯化该酶,使用该酶对人类基因组DNA进行不同温度下扩增,探究其最适反应温度,并据此对胃癌石蜡包埋组织基因组DNA进行扩增。结果:由此得到的Bst DNA聚合酶大片段能运用于胃癌石蜡包埋组织基因组DNA的扩增,扩增效率可达200倍,并能应用于a CGH芯片。结论:扩增得到保真性高,覆盖基因组范围大的DNA扩增产物。该应用与a CGH结合,使得对少量的癌症石蜡包埋组织DNA样本进行全基因组扩增,并进行其基因拷贝数变异研究成为可能。     

不同处理方式及试剂盒对石蜡包埋组织提取DNA影响

目的:比较石蜡包埋组织三种不同的前处理方式用不同试剂盒提取DNA效果,以期达到能根据各自实验室条件差异,选择合适的样本处理方式及相应的试剂盒,获得高质量DNA。方法:在提取DNA前,对石蜡块包埋的组织分别进行三种前处理,形成三种组织形式:切片机连续切片,手动刮取石蜡组织块后获得手刮片和切片后烤片制成的玻片,用Qiagen和Tiangen试剂盒提取DNA,并用琼脂糖电泳,PCR扩增和测序进行鉴定。结论:如果样本是玻片形式的石蜡包埋组织,可以选择改良过的Qiagen试剂盒提取DNA;样本如果是石蜡块包埋的组织,有切片机的,切片之后用Tiangen试剂盒提取DNA;而没有切片机的实验室可以选择用刀片刮取石蜡块组织,用Tiangen试剂盒提取DNA。     

TCR基因重排在T淋巴细胞浸润亲表皮疾病中的应用

目的:探讨具有亲表皮现象的疾病T细胞受体γ链基因重排的情况.方法:用免疫组化标记筛选17例T细胞淋巴瘤、30例可疑为T细胞淋巴瘤和10例副银屑病,采用聚合酶链式反应扩增方法检测T细胞受体γ链基因重排.结果:17例T细胞淋巴瘤中12例出现T细胞受体γ链基因重排,30例可疑为T细胞淋巴瘤和10例副银屑病均无T细胞受体γ链基因重排.结论:T细胞受体γ链基因重排检测是区分T淋巴细胞浸润的亲表皮疾病鉴别诊断的有效方法,且对T细胞淋巴瘤的确诊有重要参考价值与显著意义.     

血循环DNA在淋巴瘤中的研究进展

近年来血循环DNA用于基因诊断已成为研究热点,血循环DNA是指血浆中具有DNA双螺旋结构的核苷酸片段,逐渐成为一项新的肿瘤标记物。研究发现肿瘤患者血循环DNA较正常人有很大差异,不同疾病条件下其含量有不同程度的升高,且逐渐成为替代当前需采集肿瘤组织作为标本的无创方法。尽管血循环DNA的来源尚不清楚,通过监测血循环DNA总水平变化及相关肿瘤基因的异常改变,可以实现恶性肿瘤的早期诊断及预后评估。特别是许多国外文献报道,它与淋巴瘤的关系非常密切,无论血循环DNA的定性或定量研究,包括淋巴瘤常见的基因重排或者病毒相关血浆DNA,与淋巴瘤的诊断、治疗反应及预后直接相关。现将近几年国内外血循环DNA在淋巴瘤中的应用进行综述,对研究前景做简单展望。     

乳腺癌组织CCNA1启动子区甲基化与肿瘤转移的相关性

目的：探讨CCNA1基因甲基化在乳腺癌发生发展中的作用。方法：采用Qiagen-FFPE的步骤提取95例石蜡包埋乳腺癌组织的基因组DNA,应用限制性酶切-PCR对所提DNA进行CCNA1甲基化检测。结果：乳腺癌组织中CCNA1基因启动子甲基化的阳性率为90.9%（86/95）。结论：CCNA1基因甲基化参与乳腺癌的发生,发展,有淋巴结转移多见的特征,CCNA1基因甲基化与乳腺癌转移由关。     

Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin-fixed lymphoid tissues

Flow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin-embedded, formalin-fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin-fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the S-phase fraction was higher in paraffin-embedded tissues than in unfixed cells. This difference could be attributed to 4', 6'-diamidino-2-phenylindole dihydrochloride (DAPI), a DNA-binding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin-embedded tissues generally showed a greater S-fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of S-fraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.     

Molecular genetic analysis in the diagnosis of lymphoma in fine needle aspiration biopsies. I. Lymphomas versus benign lymphoproliferative disorders

The configurations of immunoglobulin genes, T-cell receptor (TCR) beta chain genes and bcl-2 genes were analyzed by Southern blotting in DNAs derived from 35 fine needle aspiration biopsies from various lymphoproliferative disorders. Only 1 of 16 benign lymphoproliferative disorders showed clonality: the lymph node of a patient with Wiskott-Aldrich immunodeficiency syndrome, in which clonal rearrangement of the TCR beta chain gene was detected. Clonality was demonstrated in all 14 non-Hodgkin's lymphomas (NHLs), 2 of 3 cases of Hodgkin's disease (HD) and 2 cases diagnosed as NHL or angioimmunoblastic lymphadenopathy (AILD). None of the aspirates exhibited rearrangement of the bcl-2 gene. The studies of diagnostically difficult cases proved that molecular genetic analysis of DNA, when appropriately combined with clinical data and light microscopic analysis of the lesions, can be helpful in distinguishing between: (1) a hyperplastic lymph node and NHL or AILD; (2) NHL and well-differentiated lymphocytes; and (3) a hyperplastic lymph node and HD.     

Immunoglobulin and T cell receptor gene rearrangements in lymphoproliferative disorders

Thirty-one samples representing Hodgkin's and non-Hodgkin's lymphomas, angioimmunoblastic lymphadenopathy (AILD), and benign follicular hyperplasia in HIV infections were examined for rearrangements of the immunoglobulin (Ig) and T cell receptor (TcR) beta-chain gene loci. In 11 of 12 non-Hodgkin's lymphomas (classified as Burkitt lymphoma (2), centrocytic lymphoma (1), centrocytic-centroblastic lymphoma (5), centroblastic lymphoma (3], only rearranged Ig genes could be detected. The exceptional case was an unclassified high-grade lymphoma, which represented a rearrangement of the TcR beta-chain. We also examined DNA from lymphoid neoplasms in which the lineage of the malignant cell was still controversial. Rearrangement of the TcR could exclusively be demonstrated in all 3 cases of AILD. One Ig gene rearrangement and 4 TcR beta-chain rearrangements were found in 13 samples of Hodgkin's lymphomas (11 lymph nodes, 1 pleura effusion and 1 bone biopsy with proven infiltration). Examination of 3 cases of benign follicular hyperplasia in HIV infection represented one Ig rearrangement.     

Molecular genetic analysis in the diagnosis of lymphoma in fine needle aspiration biopsies. II. Lymphomas versus nonlymphoid malignant tumors

The configurations of immunoglobulin genes and T-cell receptor beta chain genes were analyzed by Southern blotting in DNA derived from nonlymphoid malignant tumors and lymphomas. Gene rearrangements were not detected in any of the 35 cases of nonlymphoid malignant tumors. On the contrary, they were shown in all 14 cases of non-Hodgkin's lymphomas, 2 of 3 cases of Hodgkin's disease and 2 cases diagnosed as non-Hodgkin's lymphoma or angioimmunoblastic lymphadenopathy. The differentiation by light microscopy between lymphoma and nonlymphoid malignant tumors was a diagnostic problem in five cases; the molecular genetic analysis of DNA was contributory in all five diagnostically difficult aspirates. By gene rearrangement studies, the diagnosis of lymphoma was confirmed in two cases and nonlymphoid malignant tumors were accurately indicated in aspirates diagnosed finally as rhabdomyosarcoma (one case) and carcinoma (two cases).     

Diagnosis of B-cell lymphoma. Utility of the polymerase chain reaction for detecting clonality from archival cytologic smears

OBJECTIVE: To apply the polymerase chain reaction (PCR) to detect clonality for potentially helping to establish a definitive diagnosis of lymphoma in cytologic material. STUDY DESIGN: In this retrospective study, Papanicolaou-stained cytologic smears and formalin-fixed, paraffin-embedded tissues from 17 cases of B-cell lymphoma were examined to investigate their clonality by a PCR technique using three different approaches (FR3, FR3A and FR2) for amplification of immunoglobulin heavy chain genes. Cytologic smears from 10 cases of nonneoplastic lymphoid tissues and T-cell lymphomas served as negative controls. RESULTS: Monoclonality was detected in 9 of 17 cases (53%) of B-cell lymphoma in cytologic smears as compared with 8 of 16 cases (50%) in tissue sections. Semi-nested PCRs (FR3A/FR2) were superior to the single PCR (FR3) in the detection rate (41% vs. 18%). Five of seven cases (71%) of marginal zone B-cell lymphomas showed monoclonality, whereas only 4 of 10 cases (40%) of diffuse large B-cell lymphomas did so. Monoclonality was demonstrated in none of the negative controls. CONCLUSIONS: Clonality detection in B-cell lymphomas by PCR using cytologic smears is specific and equal in sensitivity to that using formalin-fixed, paraffin-embedded tissues. The detection rate is especially excellent in marginal zone B-cell lymphoma, in which the cytologic diagnosis is particularly challenging. Combined seminested PCRs for FR3A and FR2 are advocated for a reliable assessment of clonality.     

Comparison of flow cytometric data obtained using fresh and paraffin-embedded lymphoid tissue

Flow cytometric (FCM) DNA analysis was carried out on 24 lymph nodes: 13 from benign reactive hyperplasias and 11 from non-Hodgkin's lymphomas. FCM was performed on two types of samples: (1) fresh cell suspensions and (2) suspensions prepared from formalin-fixed, paraffin-embedded sections. FCM of fresh samples detected aneuploidy in 23 of the 24 cases while FCM of paraffin-embedded samples detected aneuploidy in only 6 of the 24 cases. Those six cases were lymphomas considered histologically as having a poor prognosis. Only one case, a lymphoma, was euploid with both methods. The coefficients of variance determined in each case for both methods were found to be within "normal ranges," but were greater in the paraffin-embedded specimens. The results suggest that FCM DNA analysis of formalin-fixed, paraffin-embedded sections does not have as great a resolution capacity as does analysis of fresh cell suspensions, since the former failed to detect cell populations that had a small degree of aneuploidy (close to the 2n population).     

Dual genotype in cutaneous T-cell lymphomas and pseudolymphomas

Summary Genomic DNA digests of skin biopsies from 20 patients with cutaneous T-cell lymphomas and pseudolymphomas were studied by hybridization, using probes for the constant region of the T-cell receptor beta chain and the joining region of the immunoglobin heavy chain gene. Skin biopsies from all 20 patients contained a monoclonal T-cell population. In addition, DNA from 5 patients contained an immunoglobulin gene rearrangement. These results demonstrate that cutaneous T-cell lymphomas are clonal T-cell malignancies that frequently express a dual genotype, which may sometimes reflect the clonotypic heterogeneity of these disorders.     

Mutation analyses of integrated HBV genome in hepatitis B patients

Little has been learnt in the last 30 years about detection of HBV genome as well as its mutation analysis between hepatitis B fathers （HBF） and their children. In this study, we used nest polymerase chain reaction （PCR）, fluorescence in situ hybridization （FISH）, and DNA sequencing analysis, to examine the integrated HBV genome in paraffin-embedded testis tissues, which were taken as samples from HBE and in peripheral blood mononuclear cells （PBMC） from 74 cases of HBFs and their children who were born after their fathers＇ HBV infection （caHBF）. We found that HBV DNA existed in testis tissues, mainly in the basilar parts of the seminiferous tubules, and also in PBMC of HBE It was also documented that there were point mutations of poly-loci, insertions and deletions of nucleotides in integrated HBV genomes, and the types of gene mutations in the HBFs were similar to those in caHBE This study addresses the major types of gene mutations in integrated HBV genome in human patients and also presents reliable evidence of possible genetic transmission of hepatitis B.     

Rearrangement and Reactivation of the Myelin Basic Protein Locus in Myelin-Deficient (mld) Mouse Brain

Abstract: Myelin-deficient ( mld ) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in ∼5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of ∼1.4 × 10⁻⁶ per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant ( mld/mld , scid/scid ) and single mutant ( mld/mld , + ^scid /+ ^scid ) mice, indicating that the scid factor is not required for MBP gene reactivation in mld . The significance of DNA rearrangement in mammalian development is discussed.     

Simultaneously arising Ly-1(CD5) B cell lymphomas have identical expressed IgH and kappa-genes but different nonproductive heavy chain rearrangements.

A group of CD5(Ly-1) B cell lymphomas are described. They were derived from mice which received a common pool of syngeneic mouse spleen cells. Southern blot analysis revealed that the lymphomas exhibited an unusual set of Ig gene rearrangements. Six lymphomas analyzed had either of two rearrangement patterns. EcoRI restriction digests of tumor DNA probed for rearrangements in the JH region, resulted in restriction fragments of 4.7 and 5.6 kb or of 4.7 and 8.5 kb. Each had an identical HindIII restriction fragment identified when probed for kappa gene rearrangements. Inasmuch as several B cell lymphomas from mice receiving a common pool of spleen cells had identical kappa-rearrangements and one identical IgH rearrangement, it was important to determine the DNA sequence of expressed IgH and kappa-genes. Each tumor was found to have identical nucleotide sequences of VH-DH-JH and VK-JK. The nonproductive IgH rearrangements each consisted of incomplete DH-JH rearrangements. The 8.5-kb EcoRI fragment was generated from a DFL16 gene segment rearranged into JH3, and the 5.6-kb fragment was generated from DQ52 rearranged into JH)1. We conclude that these Ly-1 B tumors are most likely derived from a single clone of cells which underwent a secondary rearrangement on the nonproductive allele after kappa-rearrangement had occurred. The alternate possibility of independently arising lymphomas with identical expressed VH and VK sequences is discussed.